The use of micro-CT in the investigation of a case involving 3D printed firearms.

This paper highlights how micro-CT was used to assist in the investigation of hybrid firearms constructed using a mixture of plastic and metal components, as a complementary technique to the physical examination performed by firearms experts. In recent years, there has been an increase in the complexity and sophistication of 3D printed and hybrid firearm designs. This was also the case in the investigation presented herein, with the firearms seized demonstrating a step change in the threat level they pose through their complexity. Thus, we describe how data produced from micro-CT scans was used to help firearms experts study the viability and mechanics of two hybrid weapons prior to dismantling and test-firing. This process aided experts in determining whether components were 3D printed or manufactured through other means, whilst ensuring that a digital record (digital twin) was retained in case evidence was damaged during testing. Finally, we show how the data was presented visually through animations and as evidence in court. This proved to be imperative when communicating to the judge, jury, and wider investigating teams, the complex multiple components and mechanisms within the firearms.

Amino acid residues in the GerAB protein important in the function and assembly of the alanine spore germination receptor of Bacillus subtilis 168.

The paradigm gerA operon is required for endospore germination in response to c-alanine as the sole germinant, and the three protein products, GerAA, GerAB, and GerAC are predicted to form a receptor complex in the spore inner membrane. GerAB shows homology to the amino acid-polyamine-organocation (APC) family of single-component transporters and is predicted to be an integral membrane protein with 10 membrane-spanning helices. Site-directed mutations were introduced into the gerAB gene at its natural location on the chromosome. Alterations to some charged or potential helix-breaking residues within membrane spans affected receptor function dramatically. In some cases, this is likely to reflect the complete loss of the GerA receptor complex, as judged by the absence of the germinant receptor protein GerAC, which suggests that the altered GerAB protein itself may be unstable or that the altered structure destabilizes the complex. Mutants that have a null phenotype for Instituto de Biotecnología de León, INBIOTEC, Parque Científico de León, Av. Real, 1, 24006 León, Spain-alanine germination but retain GerAC protein at near-normal levels are more likely to define amino acid residues of functional, rather than structural, importance. Single-amino-acid substitutions in each of the GerAB and GerAA proteins can prevent incorporation of GerAC protein into the spore; this provides strong evidence that the proteins within a specific receptor interact and that these interactions are required for receptor assembly. The lipoprotein nature of the GerAC receptor subunit is also important; an amino acid change in the prelipoprotein signal sequence in the gerAC1 mutant results in the absence of GerAC protein from the spore.

Effect of amino acid substitutions in the GerAA protein on the function of the alanine-responsive germinant receptor of Bacillus subtilis spores.

Spores of Bacillus subtilis require the GerAA, GerAB, and GerAC receptor proteins for L-alanine-induced germination. Mutations in gerAA, both random and site directed, result in phenotypes that identify amino acid residues important for receptor function in broad terms. They highlight the functional importance of two regions in the central, integral membrane domain of GerAA. A P324S substitution in the first residue of a conserved PFPP motif results in a 10-fold increase in a spore's sensitivity to alanine; a P326S change results in the release of phase-dark spores, in which the receptor may be in an "activated" or "quasigerminated" state. Substitutions in residues 398 to 400, in a short loop between the last two likely membrane-spanning helices of this central domain, all affect the germination response, with the G398S substitution causing a temperature-sensitive defect. In others, there are wider effects on the receptor: if alanine is substituted for conserved residue N146, H304, or E330, a severe defect in L-alanine germination results. This correlates with the absence of GerAC, suggesting that the assembly or stability of the entire receptor complex has been compromised by the defect in GerAA. In contrast, severely germination-defective mutants such as E129K, L373F, S400F, and M409N mutants retain GerAC at normal levels, suggesting more local and specific effects on the function of GerAA itself. Further interpretation will depend on progress in structural analysis of the receptor proteins.

Spore Germination.

Despite being resistant to a variety of environmental insults, the bacterial endospore can sense the presence of small molecules and respond by germinating, losing the specialized structures of the dormant spore, and resuming active metabolism, before outgrowing into vegetative cells. Our current level of understanding of the spore germination process in bacilli and clostridia is reviewed, with particular emphasis on the germinant receptors characterized in Bacillus subtilis, Bacillus cereus, and Bacillus anthracis. The recent evidence for a local clustering of receptors in a "germinosome" would begin to explain how signals from different receptors could be integrated. The SpoVA proteins, involved in the uptake of Ca2+-dipicolinic acid into the forespore during sporulation, are also responsible for its release during germination. Lytic enzymes SleB and CwlJ, found in bacilli and some clostridia, hydrolyze the spore cortex: other clostridia use SleC for this purpose. With genome sequencing has come the appreciation that there is considerable diversity in the setting for the germination machinery between bacilli and clostridia.